In the past decade, a great deal of progress has been made in the field of targeted genomic engineering. Technologies such as designer zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and homing meganucleases have made possible site-specific genome modifications in many different model organisms, ranging from zebrafish to mammalian cells. Based on the results to date, however, genome editing tools that are efficient, flexible, and cost-effective have remained elusive.
Small RNA-based defense systems that provide adaptive, heritable immunity against viruses, plasmids, and other mobile genetic elements have been identified in archaea and bacteria. The discovery of the prokaryotic type II CRISPR (clustered regularly interspaced short palindromic repeats) system, originally identified in the bacterium Streptococcus pyogenes as a mechanism to defend against viruses and foreign DNA, where prokaryotes with CRISPR-Cas immune systems capture short fragments of invader genetic material with the CRISPR loci in the genomes, and small RNAs produced from the CRISPR loci (crRNAs) guide Cas proteins to recognize and degrade the invading nucleic acids. It has been proposed that the bacterial CRISPR-Cas system has the potential to provide a tool for targeted genome engineering. However, the CRISPR system is taken from prokaryotes, and adapting those bacterial components to operate in mammalian host cells in an efficient, rapid, adaptable and cost-effective manner remains unproven.
In a native prokaryotic host, the CRISPR/CRISPR-associated (Cas) system involves (1) integration of short regions of genetic material that are homologous to the foreign invading nucleic acids, called “spacers”, in clustered arrays in the host genome, (2) expression of short guiding RNAs (crRNAs) from the spacers, (3) binding of the crRNAs to specific portions of the foreign DNA called protospacers, and (4) degradation of protospacers by CRISPR-associated nucleases (Cas). A Type-II CRISPR system has been previously described in the bacterium Streptococcus pyogenes, where four genes (Cas9, Cas1, Cas2, Csn1) and two non-coding small RNAs (pre-crRNA and tracrRNA (trans-activating CRISPR RNA)) act in concert to target and degrade foreign DNA in a sequence-specific manner (Jinek et al., “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity,” Science 337(6096):816-821 (August 2012, epub Jun. 28, 2012)). Cas9, is a double stranded nuclease, with two active cutting sites, one for each strand of the double helix. Cas9 is thought to be the only protein involved in the crRNA/tracrRNA-guided silencing of foreign nucleic acids.
The specificity of binding of the CRISPR components to the foreign DNA is controlled by the non-repetitive spacer elements in the pre-crRNA, which upon transcription along with the tracrRNA, directs the Cas9 nuclease to the protospacer:crRNA heteroduplex and induces double-strand breakage (DSB) formation. Additionally, the Cas9 nuclease cuts the DNA only if a protospacer adjacent motif (PAM) nucleotide sequence is present immediately downstream of the protospacer sequence, whose canonical sequence in S. pyogenes is 5′-NGG-3′, where N refers to any nucleotide.
The potential for designing a customizable RNA-programmed CRISPR/Cas9 system that would function in mammalian cells has been explored, with the hope that such systems would provide customizable genome-editing applications in mammalian cells. Cong et al., “Multiplex Genome Engineering Using CRISPR/Cas Systems” Science 339(6121):819-823 (Feb. 15, 2013), epub Science Express (Jan. 3, 2013); Mali et al., “RNA-Guided Human Genome Engineering via Cas9” Science 339(6121):823-826 (Feb. 15, 2013), epub Science Express (Jan. 3, 2013); and Cho et al., “Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease,” Nature Biotechnology 31:230-232 (epub Jan. 29, 2013). Various groups have introduced double stranded breaks at endogenous genomic loci in human cells and mouse cells using a codon-optimized version of the S. pyogenes Cas9 protein. These systems incorporate and express a single engineered chimeric crRNA:tracrRNA transcript (which would normally be expressed as two different RNAs in the native type II CRISPR bacterial system), and where the chimeric transcript further contains targeting sequence to a desired genomic target. These crRNA-tracrRNA fusions are alternatively termed guide RNA, chimeric guide RNA or targeting RNA. These components are sufficient in mammalian host cells to direct the double stranded cleavage of a target genomic DNA in a sequence-specific manner.
Mutant forms of Cas9 nuclease have been developed to take advantage of the Cas9 nuclease activity in genome engineering and transcriptional regulation. One mutant form of Cas9 nuclease containing a single amino acid substitution (D10A) has lost its native double-stranded nuclease activity present in the wild type form, but retains partial function as a single-stranded nickase (Jinek et al., Science 337(6096), p. 816-821 [August 2012], epub Jun. 28, 2012), generating a break in the complementary strand of DNA rather than both strands as with the wild-type. This allows repair of the DNA template using a high-fidelity pathway rather than non-homologous end joining (NHEJ). The higher fidelity pathway prevents formation of indels at the targeted locus, and possibly other locations in the genome to reduce possible off-target/toxicity effects while maintaining ability to undergo homologous recombination (Cong et al., Science 339(6121):819-823 (Feb. 15, 2013), epub Science Express (Jan. 3, 2013)). Paired nicking has been shown to reduce off-target activity by 50- to 1,500 fold in cell lines and to facilitate gene knockouts in mouse zygotes without losing on-target cleavage efficiency (Ran et al., “Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity.” Cell 154(6):1380-1389 (September 2013)). A double-mutant (DM) form of Cas9 containing the D10A mutation as well as a H840A mutation shows no single-stranded or double-stranded nuclease activity, and is termed a null nuclease.
Precise editing or replacement of mutant genes and introduction of new gene sequences into cells has remained a goal of gene therapy since its inception. The ability to edit chromosomal DNA and regulate transcription in vivo using customizable gene-specific tools remain a powerful tools in the study of biological systems and holds promise for the study of cellular systems, human physiology and a potential therapeutics in the treatment of human disease. Gene regulatory tools can be used, for example, to study biology by perturbing gene networks, and also for example, can be used to treat genetic diseases by repairing genetic defects or by introducing genes into cells for therapeutic purposes.
This goal looked promising when it was demonstrated that targeted cleavage of chromosomal sequences and enhanced homologous recombination (HR) could be achieved using chimeric molecules composed of a nuclease domain and customizable reprogrammed DNA-recognition domains, including zinc-finger nucleases (ZFNs) and transcription activator-like (TAL) effector nucleases (TALENs). However, these two systems face technical and practical limitations, including off-site cleavage events, cell toxicity, and are time and cost intensive to develop.
The potential for using CRISPR/Cas9 components to construct customizable systems to regulate transcription has been proposed. It has been demonstrated that a catalytically silent Cas-9 mutant (a null nuclease) can be tethered to specified gene promoter regions and has the effect of reducing expression of those genes. These systems are limited in their utility due to the slight gene suppression effects that can be achieved.
The possibility of constructing chimeric transcription factors to regulate gene expression using the DNA targeting ability of CRISPR/Cas9 components combined with tethered transcriptional activation or suppression domains has been suggested. See Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell 154(2):442-451 (epub Jul. 11, 2013); Perez-Pinera et al, “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,” Nature Methods 10:973-976 (epub Jul. 25, 2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nature Biotechnology 31:833-838 (epub Aug. 1, 2013). Optimized forms of these CRISPR/Cas chimeric transcription factors have yet to be developed.
Genome engineering and genetic modulation by the control of individual gene expression hold great promise for research and potential therapeutics. CRISPR/Cas RNA-guided genome targeting and gene regulation in mammalian cells using modified bacterial CRISPR/Cas components defines a potential new class of tools with broad applicability to diverse fields in biology and medicine. However, the development of such tools must be improved in order to make these modalities more cost effective and less time consuming.
What is needed in the art are rapid, cost-effective and versatile tools and methods for RNA-programmed genome engineering and RNA-programmed control of gene expression. Ideally, these tools will have an ease in designing sequence guide molecules and also show a high efficiency of delivery into host cells such as mammalian cells. The development of systems that use small RNAs as guides (such as CRISPR/Cas systems) to cleave DNA in a sequence-specific manner resulting in directed genomic modification or to synthesize customized transcription factors for a particular gene of interest will find significant utility.
The present invention, in its many embodiments, provides compositions and methods that overcome these challenges in the industry, have a number of advantages over the state of the art and provide many benefits previously unrealized in other types of products. In addition, still further benefits flow from the invention described herein, as will be apparent upon reading the present disclosure.